Pressure-controlled nucleic acid hybridization

ABSTRACT

A method of hybridizing a first nucleic acid to a second nucleic acid at least partially complementary to the first nucleic acid by (1) providing a sample vessel and pressure controller for the vessel; and (2) contacting the first and second nucleic acids within the vessel at a pressure above ambient pressure that is effective to enhance hybridization of the first and second nucleic acids.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/076,478, entitled “Pressure-Controlled Nucleic AcidHybridization”, Laugharn et al., filed on Mar. 2, 1998, now abandoned;and is a continuation-in-part International Application PCT US97/11198,with an international filing date of July 1, 1997, now abandoned.

FIELD OF THE INVENTION

The invention is in the general field of nucleic acid hybridization.

BACKGROUND OF THE INVENTION

Nucleic acid hybridization is an important technique for detecting thepresence of particular sequence information. Recent advances haveallowed for the production of high density oligonucleotide arrays whichmay have great utility for research and clinical diagnostics. Typically,a sequence is amplified and labeled with fluorescent tags. The sample isincubated with the probe array and the sample is washed with a series ofincreasingly stringent buffers. Stringency may be provided by means ofreduced salt concentration or increased temperature. Reduced saltconcentrations lead to electrostatic repulsion between phosphate groups,thereby lowering the melting temperature (T_(m)).

The number of different sequences on each array is ultimately limited bythe detection capabilities of the instrument analyzing it and factorssuch as contaminating fluorescent compounds, stray light, andhybridization of mismatched oligonucleotides. As the stringency ofhybridization is increased, the noise level from mismatches is reduced(increasing specificity), but the overall signal is also reduced due toloss of correctly hybridized molecules (decreasing sensitivity).

Two related and important amplification techniques which depend on thespecificity and sensitivity of oligonucleotide hybridization arepolymerase chain reaction (PCR) and ligase chain reaction (LCR), whichare commonly used in medical diagnostics and research.

SUMMARY OF THE INVENTION

The invention relates to controlling the specificity, sensitivity, orselectivity of nucleic acid hybridization procedures. Control ofhybridization according to the present invention is achieved throughapplication of high hydrostatic pressure. Without intending to belimited to any mechanism, it is believed that increased pressure favorsnucleic acid hybridization (i.e., reversible or irreversiblehybridization). Regardless of the mechanism involved, high pressureincreases the sensitivity, specificity, or selectivity of nucleic acidhybridizations.

Accordingly, the invention features a method of hybridizing a firstnucleic acid to a second nucleic acid at least partially complementaryto the first nucleic acid by (1) providing a sample vessel and pressurecontroller for the vessel; and (2) contacting the first and secondnucleic acids within the vessel at a pressure above ambient pressure(e.g., above 10,000 psi) which is effective to enhance hybridization ofthe first and second nucleic acids. This method opitionally includescycling pressure in the vessel between a first higher pressure at whichthe first and second nucleic acid are hybridized and a second lowerpressure at which the first and second nucleic acid are denatured. Incase of pressure cycling, can further include providing a temperaturecontrol for the sample vessel, and cycling the temperature between alower temperature and a higher temperature, such that the first andsecond nucleic acids hybridize at the first pressure and lowertemperature, and such that the first and second nucleic acids denatureat the second pressure and higher temperature. Alternatively, the vesselcan be maintained at a constant temperature as the pressure is cycled.Such methods can be used to amplify a portion of the second nucleicacid. An optional step in any of the above methods includes washing awayunhybridized nucleic acids after increasing the pressure but beforedecreasing the pressure.

In another embodiment, the invention features a method of detecting in asample the presence of a nucleic acid that hybridizes to a referencenucleic acid at a first higher pressure but not at a second lowerpressure by (1) providing a sample vessel and pressure controller forthe vessel; and in any order (2) contacting the reference sequence withthe sample in the vessel at the first pressure; (3) contacting thereference sequence with the sample in the pressure vessel at the secondpressure; and (4) detecting the presence of a nucleic acid thathybridizes to the reference nucleic acid at the first pressure but notat the second pressure. In one aspect, the reference sequence is firstcontacted with the sample and hybridization is detected, and then thepressure is lowered and the absence of hybridization is detected.

The invention also features a method of discriminating between a firstnucleic acid and a second nucleic acid that is different from the firstnucleic acid by (1) providing a sample vessel and pressure controllerfor the vessel; (2) maintaining the vessel at a constant pressure; (3)providing the first and second nucleic acid and a reference nucleic acidin the vessel under conditions that do not allow either the first or thesecond nucleic acid to hybridize to the reference nucleic acid; (4)perturbing at least one condition (e.g., temperature or an electricfield) to establish conditions that permit the first nucleic acid toform a complex with the reference nucleic acid at equilibrium and topermit the second nucleic acid to form a complex with the referencenucleic acid at equilibrium; and (5) comparing the time necessary toachieve equilibrium hybridization between the first nucleic acid and thereference nucleic acid with the time necessary to achieve equilibriumhybridization between the second nucleic acid and the reference nucleicacid, wherein the difference indicates the relative difference insequence between the first and the second nucleic acids.

In addition, the invention features a method of discriminating between afirst nucleic acid and a second nucleic acid that is different from thefirst nucleic acid by (1) providing a sample vessel and pressurecontroller for the vessel; (3) providing the first and second nucleicacid and a reference nucleic acid in the vessel under a first pressurethat does not allow either the first or the second nucleic acid tohybridize to the reference nucleic acid; (4) perturbing the pressure toestablish conditions that permit the first nucleic acid to form acomplex with the reference nucleic acid at equilibrium and to permit thesecond nucleic acid to form a complex with the reference nucleic acid atequilibrium; and (5) comparing the time necessary to achieve equilibriumhybridization between the first nucleic acid and the reference nucleicacid with the time necessary to achieve equilibrium hybridizationbetween the second nucleic acid and the reference nucleic acid, whereinthe difference indicates the relative difference in sequence between thefirst and the second nucleic acids.

Methods of placing nucleic acids and optionally enzymes under pressureare described in WO 96/27432.

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “solution” refers to a liquid, and moreparticularly, the incorporation of substances (e.g. dissolved compounds)in a liquid. The term “aqueous solution” refers to a solution eithercontaining water or that is like water. For example, the presentinvention contemplates the use of assay buffers that are aqueoussolutions.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” The term“expression vector” as used herein refers to a recombinant DNA moleculecontaining a desired coding sequence and appropriate nucleic acidsequences necessary for the expression of the operably linked codingsequence in a particular host organism. Nucleic acid sequences necessaryfor expression in prokaryotes usually include a promoter, an operator(optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.” Complementarity maybe “partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods which depend upon binding between nucleicacids.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41 (% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. With “high stringency” conditions, nucleicacid base pairing will occur only between nucleic acid fragments thathave a high frequency of complementary base sequences. Thus, conditionsof “weak” or “low” stringency are often required with nucleic acids thatare derived from organisms that are genetically diverse, as thefrequency of complementary sequences is usually less.

As used herein, the terms “nucleic acid” and “nucleic acid substrate”encompass both DNA and RNA, whether single or double stranded. It is notintended that the present invention be limited by the length of thenucleic acid; the nucleic acid may be genomic or a defined length (e.g.short oligonucleotides) or fragments thereof (including single bases).It is also not intended that the present invention be limited by thenature or source of the nucleic acid. It may be naturally occurring,purified, produced synthetically, recombinantly or by amplification. Theterm “modified nucleic acid substrate” refers to the alteration of thestructure of the nucleic acid substrate. To illustrate, an enzyme mayremove a nucleotide from the nucleic acid substrate, yielding a modifiednucleic acid substrate.

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids which may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample. In contrast, “background template” is used inreference to nucleic acid other than sample template which may or maynot be present in a sample. Background template is most ofteninadvertent. It may be the result of carryover, or it may be due to thepresence of nucleic acid contaminants sought to be purified away fromthe sample. For example, nucleic acids from organisms other than thoseto be detected may be present as background in a test sample.

As used herein, the term “sample” is used in its broadest sense. Thatis, the term may encompass a specimen, culture, biological sample,environmental sample, etc. The term includes human and animal samples aswell as naturally occurring and synthetic material. The term “samplevessel” is used to indicate a means for containing a sample, whether byenclosing a sample (e.g., in a batch format) or by using a sample in anenclosed device (e.g., channeling both within and between a chamber,channel or stream). Similarly, a “reaction vessel” is not limited to anyone design; typically it is a sample vessel in which a reaction takesplace.

A “reaction mixture” refers to the mixing together of two or morecomponents such that a reaction will occur. The term “optimum enzymatictemperatures” refers to those temperatures at which an enzyme is mostactive. Thus, it depends on the characteristics of the individualenzyme. Typically, the optimum is between approximately 10 andapproximately 80 degrees Centigrade, and more particularly betweenapproximately 25 and 37 degrees Centigrade.

As used herein, the term “inhibition” of enzyme activity be pressurerefers to an enzyme that is reversibly inhibited at a particularpressure. When the pressure is altered (for example, lowered), theenzyme resumes its level of original activity.

As used herein the terms “substantially inactive” and “renderingsubstantially inactive” refer to an enzyme that exhibits less thanapproximately 20%, and generally less than 10%, of its activity atoptimum enzymatic temperature (100% activity). An enzyme that has beenrendered substantially inactive may also exhibit no activity, but may bereversibly inhibited such that activity of approximately normal levelsreturns under appropriate conditions of pressure, temperature and thelike.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension produce which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, which is capable ofhybridizing to another oligonucleotide of interest. Probes are useful inthe detection, identification and isolation of particular genesequences. It is contemplated that any probe used in the presentinvention will be labelled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is furthercontemplated that the oligonucleotide of interest (i.e., to be detected)will be labelled with a reporter molecule. It is also contemplated thatboth the probe and oligonucleotide of interest will be labelled. It isnot intended that the present invention be limited to any particulardetection system or label.

As used herein, the term “target” refers to the region of nucleic acidbounded by the primers used for detection and/or amplification (e.g., bythe polymerase chain reaction). Thus, the “target” is sought to besorted out from other nucleic acid sequences. A “segment” is defined asa region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202,hereby incorporated by reference, which describe a method for increasingthe concentration of a segment of a target sequence in a mixture ofgenomic DNA without cloning or purification.

As used herein, the terms “PCR product” and “amplification product”refer to the resultant mixture of compounds after two or more cycles ofthe PCR steps of denaturation, annealing and extension are complete.These terms encompass the case where there has been amplification of oneor more segments of one or more target sequences.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleoside triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.) As used herein, the terms “restrictionendonucleases” and “restriction enzymes” refer to bacterial enzymes,each of which cut double-stranded DNA at or near a specific nucleotidesequence.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule which is comprised of segments of DNA joinedtogether by means of molecular biological techniques.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides referred to as the “5′end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5′ orupstream of the coding region. However, enhancer elements can exerttheir effect even when located 3′ of the promoter element and the codingregion. Transcription termination and polyadenylation signals arelocated 3′ or downstream of the coding region.

As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a DNA sequence comprising the codingregion of a gene or in other words the DNA sequence which encodes a geneproduct. The coding region may be present in either a cDNA or genomicDNA form. Suitable control elements such as enhancers/promoters, splicejunctions, polyadenylation signals, etc., may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc., or a combination of both endogenous andexogenous control elements.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

Enzymes that synthesize polymers may dissociate after each catalyticevent, i.e., they may be “nonprocessive.” On the other hand, they mayremain bound to the polymer until many cycles of reaction are completed,i.e., they may be “processive.” See A. Kornberg, DNA Replication(Freeman and Co. 1980)

Other features or advantages of the present invention will be apparentfrom the following detailed description and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the realization that the molar volumes ofmismatched nucleic acid hybridization products are larger than the molarvolumes for complementary hybridization products. Thus, high pressurewill tend to drive the hybridization reaction equilibrium towardscorrect matches and away from mismatches and result in a higher signalto noise ratio in a given hybridization. Higher temperatures may also beemployed for a further decrease in mismatching. Dissociation of fullycomplementary nucleic acids can be slowed by high pressure, whereasdissociation of mismatched sequences can be enhanced or slowed to alesser degree than fully complementary hybrids. Pressure effects onmelting and dissociation may provide information that allows assignmentof a mismatched base with fewer than four probes or may increases thesensitivity of a hybridization reaction.

Modulation of hydrostatic pressure has multiple advantages inhybridization assays. Pressure allows rapid modulation of the stringencyof hybridization. By raising the melting temperature of hybrids, higherhybridization temperatures may be used with the advantage of more rapidapproach to equilibrium. Pressure differs from other forms of stringencymodulation such as salt concentration or temperature in that it providesadditional discrimination between match and mismatch. This is ofparticular advantage when the base to be discriminated is at or near theterminus of one of the sequences to be assayed. The additionalsensitivity provided by increased specificity under pressure allows formultiple benefits, such as more rapid data collection, higher densityarrays, and shorter hybridization times. Pressure operates withoutexchange of fluids and may be transmitted at up to the speed of sound.

Several devices may be used for the application of hydrostatic pressureto hybridization reactions, arrays or microchip-based hybridizationarrays. The reaction may be sealed in a deformable container and thecontainer is placed in a device that generates elevated hydrostaticpressures and is temperature controlled. After the hyperbarichybridization reaction is complete, the reaction is removed from thepressurizing apparatus and analyzed by the various means known in theart.

Another useful device is a hyperbaric chamber with an optical window.Such devices are known in the art of high pressure science and includethe diamond anvil cell, the sapphire anvil cell and fiber-opticfeed-throughs into hyperbaric chambers (Eremets, M “High PressureExperimental Methods”, 1996, Oxford University Press: New York). Withthis device, the effects of changing the pressure or temperature may bemeasured continuously. For example, the rate of strand dissociation in ahybridization array may be measured after a rapid pressure drop to yieldsequence information. This experiment may make use of the knowledge thatpressure reduces the rate of hybrid melting so that by raising thepressure and then the temperature, one can put the system in a state ofnon-equilibrium which is high in potential energy. The pressure is thenreleased and the rate of strand melting is measured.

Alternatively, a hybridization reaction may be conducted at loweffective stringency by being conducted at high pressure. The pattern ofhybridization to a defined polynucleotide array is determined by opticalor other means. The pressure is then reduced to lower the stringency,and the change in the pattern of bound DNA is again determined,optionally after flushing away nucleotides which were released. Furthercycles of pressure lowering and optional flushing allow determination ofthe degree of affinity of the bound DNA to the array.

In a method of the invention, the hybridization reaction is allowed tocome to equilibrium while under stringent conditions at an elevatedhydrostatic pressure, such as 100,000 psi. The reaction is then washedwith a mildly stringent buffer and the hybridization pattern isdetermined.

In another method of the invention, the hybridization reaction occursunder conditions of low stringency and then is washed under conditionsof high stringency while under pressure. A device suitable for thispurpose is (“Version3”) described in WO 96/27432. Other devices such asthose utilizing electrophoresis or electroosmosis may be used. Thewashing solution may be held under pressure and then rapidly replacedwith a non-stringent solution at atmospheric pressure, provided theexchange is rapid enough that the system does not significantly returnto a condition of reduced selectivity.

The extent of sequence-specific hybridization is determined bymeasurement of the rate of relaxation toward equilibrium following aperturbation such as a rapid temperature jump at several hydrostaticpressures. The pressure effect on the rate of approach to equilibriumwill be a sensitive discriminator of matched vs. mismatched sequencesand will allow for a well-defined and more easily measured dissociationrate. In addition, singly and multiply mismatched sequences will be moreeasily determined. The measured dissociation rate at high pressure maybe sufficient to accurately discriminate between mismatches.

Another perturbation which may be used in this technique is electricalperturbation as described in U.S. Pat. Nos. 5,632,957 and 5,605,662 andinvolves monitoring changes in fluorescence while short pulses ofelectric field perturbations are applied.

Since the methods of the invention results in increased specificity,sensitivity, or selectivity of hybridization, such methods can be usedto reduce mis-primings in technological applications involvingpolymerase or ligase enzymes, which require hybridization of a primer toa template. Examples include PCR, Sanger sequencing reactions, and LCR.Hybridizations may be done under high pressures such as 10,000 psi or100,000 psi. It is possible to find a pressure at which the optimalhybridization temperature is the same as the optimal temperature forpolymerase extension, thereby decreasing the number of thermal cyclingsteps needed for any suitable application. In addition, it isanticipated that fewer errors will result in polymerase extensionreactions at elevated pressures the specificity of the editing mechanismis increased due to increased exonuclease activity. Optimal pressuresfor hybridization and extension steps may differ and are expected to bein the range of 5000 psi to 100,000 psi. High pressures have the addedadvantage of further stabilizing the thermostable enzymes which areoften used in these reactions.

Improved Biochip Array Performance

An important application for the techniques and apparatus of theinvention is in the improved speed and accuracy of readout in“sequencing by hybridization” and in the closely related,currently-commercial techniques applied to gene identification anddiagnosis. In these techniques, an array of single-stranded nucleicacids—usually DNA, or less commonly RNA, and in principle includingnon-natural nucleic acids such as “peptide” nucleic acids—is synthesizedon the surface of a “chip”, of silicon, silica/glass, or other planarmaterial. Techniques derived from microelectronics—in particular,sequential use of masks followed by chemical development, sometimesoptically stimulated—allow the simultaneous synthesis of differingsequences of nucleic acids in different areas of the same chip. (Theprinciples are extensively described in the literature, and are known inthe art.) The resultant pattern can be used for gene diagnosis, or forsequencing of DNA.

To use the nucleic acid chips, the nucleic acids to be tested arelabeled in any known manner, typically with one or more fluorescentlabels. The nucleic acids are then rendered single-stranded, ifnecessary, and allowed to hybridize with the single stranded nucleicacids on the chip. The pattern of hybridization allows at least partialdetermination of the sequence of the tested nucleic acid, since thesequence of the areas of the chip to which the tested nucleic acidshybridized are known.

However, mismatches during hybridization are possible. For example, achip sequence of 8 nucleotides will bind tested sequences in which 8, 7,6, or even 5 or less of the bases within the nucleic acid are properlybase-paired, depending on the “stringency” of the hybridizationconditions. The concept of “stringency” is well-known in the art. Inessence, when the conditions in the test solution (e.g., saltconcentration, buffer composition, pH and temperature) favor binding,the hybridization conditions are described as “less stringent” or “oflow stringency”. When conditions are less favorable to hybridization,then only well-matched sequences will hybridize, and the conditions aredescribed as “stringent”, or “more stringent”. Under ideal stringency,only the target sequence of interest (e.g., a perfectly complimentarysequence) will hybridize to a reference nucleic acid.

In diagnosis of genetic shifts, if a sample of DNA is restricted,labeled, and rendered single-stranded, then with an ordered array ofnucleic acids on the test chip, and at high stringency, only sequencesspecific to the various alleles of the gene in question will bind.However, in actual situations, and in particular when amplificationmeans are not used, many different restriction fragments of the genomewill have some affinity for the array, and at low stringency, binding oftest nucleic acid to most portions of the array will occur. On the otherhand, at high stringency, hybridization will be slow even forwell-matched nucleic acids, and may tend to be incomplete, resulting inloss of signal.

Therefore, as a practical matter, such tests are usually initiated atlow stringency, where reactions are fast and quantitative, and then thestringency is gradually increased by increased temperature and/or lowerionic strength, and at the end of the process only highly homologousnucleic acids are still hybridized to the chip. The last nucleic acidsto dissociate are normally the best matched to that particular area ofthe chip, and therefore are judged to be substantially similar to thetarget sequence.

While these procedures can be effective, they are also slow. Withtypicaly hybridization times of many minutes for each step of increasedstringency, arriving at optimal conditions can take up to a few hours.The essence of the invention is the application of control ofhybridization stringency by pressure. As noted above, pressure changesare very rapid, and have a characteristic time of equilibration of lessthan a microsecond in small volumes. One can thus arrange a continuousor pulsed flow of buffer of constant composition, and conduct thestringency analysis by decreasing the pressure. The speed of successivesteps of increased stringency is limited only by the time required forless stringently bound nucleic acids to dissociate from the referencenucleic acid. Moreover, the speed of hybridization is increased athigher pressure, so the initial low-stringency hybridization step isalso faster. As noted above, the time required for dehybridization, aswell as any optical or other signals generated by dehybridization, mayalso be a factor in sequence determination.

Because the capital cost of the sequencing-by-hybridization equipment isa significant portion of the cost of performing such an assay, the useof pressure changes to control hybridization (in place of the usualtemperature and/or buffer change) can increase the speed and accuracy ofthe analysis, which in turn can significantly lower the cost of suchassays.

A system to perform such pressure-hybridization assays is also a part ofthe invention. The mechanical portions of such a device have previouslybeen described in WO 96/27432. The device includes a pressurizablechamber with means for rapid pressure changes between atmospheric and ahigher pressure (such as 5,000, 10,000, or 50,000 psi), and means forintroduction and removal of fluids. The device is adapted to improvesequencing-by-hybridization by inclusion of a chip or slide bearingdefined immobilized nucleic acid sequences in known areas, and a flowarrangement which efficiently washes the nucleic acid-bearing surface(s)of the chip with a buffer, to remove unhybridized nucleic acids, and ameans for detecting the extent of dehybridization and the pattern ofremaining hybridization on the chip.

The detection means may be any means known in the art which arecompatible with the high pressures and sudden pressure changes necessaryfor the assays. The currently-preferred means for detection ofhybridized test DNA is a fluorescent label, and the detection isaccomplished by direct visualization. To use this system, thepressurizable chamber is provided with an optical window which allowsaccurate projection of emitted fluorescent light onto a detection means,such as a video camera or a photodiode array. A suitable means is animaging lens embedded in the wall or end of the pressurizable reactionvolume, which images the surface of the chip to an outside detectionmeans. An intermediate adaptable lens, with position and/or opticalpower controlled electronically, and preferably located outside thepressurizable zone, can be used to correct distortions arising fromchanges in pressure on the imaging lens. Another option is a flatsapphire window, with an adjustably-positioned detection device, as isknown from the literature related to the analytical ultracentrifuge.

Alternatively, the detection means could be a light-sensitive diodearray mounted behind the nucleic acid array-bearing chip, baffled sothat fluorescent light emitted from a defined area of the array falls ona particular diode or diodes, with a suitable pressure-resistant cablefor conduction signals to an outside display and recording means, suchas a computer.

As noted above, the effects of pressure will be beneficial in performingvarious sorts of nucleic acid amplification assays. These are describedhere in terms of some well-known DNA amplification methods, and are alsoapplicable to amplification of other types of nucleic acids.

Nucleic Acid Amplification under High Pressure

Polymerase Chain Reaction (PCR). In this widely practiced method, aparticular sequence of DNA, typically a restriction fragment, isamplified by use of a primer, or preferably two primers, and a DNApolymerase, in the presence of four deoxyribonucleoside triphosphates(dNTPs). The primer is allowed to hybridize, at a firsthybridization-permissive temperature, to a stretch of ssDNA to beamplified. Then the polymerase, which requires a short sequence ofdouble-stranded DNA as a “start” point, will proceed along the DNA tomake a copy of it. If there are two primers, then amplification proceedson both strands. Next, the reaction mixture is heated to dissociate thenewly-formed copies from the original. On cooling, new primershybridize, and the cycle is repeated. In principle, the numbers ofcopies of the target DNA is doubled in each cycle, leading to a largeincrease in the amount of the target DNA sequence.

The amplified copies can be fluorescently labelled. If di-deoxynucleotides are used in the last few cycles, then the resultant mixturecan be used for Sanger-type sequencing.

High pressure offers three distinct avenues for improvement of the PCR.

First, it can be used to replace the temperature-cycling step with apressure controlled dehybridization/hybridization cycle. This isinherently faster and allows a “quick-start” procedure. In thehigh-pressure-PCR (HP-PCR) assay, the polymerase's sensitivity topressure is determined, and a first pressure P3 is selected. P3 is lowenough to retain enzymic activity and high enough to enable primerbinding. A temperature T1 is selected at which the enzyme can function,and at which the primers (and primer extension products) will bedehybridized from the test DNA at low pressure. Typically, enzymes thatare thermostable have enhanced tolerance to hyperbaric conditions. Thistemperature (T1) can be predicted from first principles and the primersequence composition, and quickly optimized to the actual situation.

The reaction mixture is prepared at an initial P1/T1 state, except forthe polymerase, and is raised to the P3/T1 state. The primers hybridizeto the template. Enzyme is added under pressure and the primers areextended at a P2/T1, where P2 can be between P1 and P3. After the newstrands have been polymerized, the pressure is dropped to P1 at whichthe primers and extension products will dehybridize. The pressure israised back to P3/T1 and held briefly to allow primerhybridization/extension before progressing to P2/T1 for primerextension. Then pressure is reduced to P1/T1, and the cycle is repeated.The consequences at the P1/T1, P2/T1, and P3/T1 states are summarizedbelow.

P1/T1 P2/T1 P3/T1 Enzyme active Yes Yes Yes Primer & template bind YesNo Yes Primer & template denature Yes No No Primer extends No Yes N/ADouble-strand denatures Yes No No

The reactions immediately above were conducted at one temperature, T1,to illustrate one example of the methods of this invention. However,performing reactions at different temperatures in conjunction withdifferent pressures can be ideal under some circumstances.

Because pressure changes propagate through the medium at the speed ofsound, and require no convection or mixing, pressure changes can occurthroughout a medium much quicker than temperature changes. Consequently,the reaction medium spends more time at equilibrium. Therefore, the timerequired to conduct a PCR reaction is reduced. Since PCR is important ingenome sequencing, and increasing in forensics and diagnostics, thedecrease in time required is advantageous.

Second, careful selection of temperatures and pressures can increase theperformance of an enzyme. As noted above, in many cases it will bepossible to find a temperature and pressure combination at which theactivity of the enzyme is optimized, which will shorten the timerequired in the polymerization step. This speed improvement isindependent of the speed improvement obtained by pressure changes. Inaddition, by selection of temperature and pressure, the stability of theenzyme against denaturation or other inactivation can be enhanced. Inparticular, the same optimum pressure for activity will normally sufficeto prevent enzyme denaturation, because the additional cycling stepinvolves a decrease in pressure (disfavoring enzyme denaturation) ratherthan an increase in temperature (favoring enzyme denaturation). This mayallow additional kinds of polymerases to be used in PCR.

Moreover, at some combinations of temperature and pressure, thepolymerase will be more tightly bound to the DNA being replicated, andis expected to exhibit increased processivity, resulting in longerstrands on average, and a higher proportion of fully replicated strandsin the mixture. This is critical when using PCR to amplify longstretches of DNA, for example 1000 bases or more.

Third, running the PCR at high pressure is especially beneficial becausethe effective stringency of the reaction can be improved at highpressure. Improvement of stringency is described above in the discussionof “DNA chips”. Because pressure widens the temperature spread betweenhybridization of matched and mismatched primers, binding of primers tomismatched sites can be significantly reduced at high pressure comparedto ambient pressure. The speed of change between dehybridizing andhybridizing conditions can also diminish the proportion of mismatchedhybridizations in the mixture. In turn, there will be fewer errors inthe amplified sequences, resulting in better quality (via higherfidelity of polymerization) of the amplified DNA for its intended use,such as for sequencing, or as a probe for use in a reaction involving aDNA chip.

In addition, the primers can be shorter in a high-pressure reaction,because formation of double-stranded regions is favored at highpressure. Current practice favors longer primers both for specificityand sensitivity of hybridization. With pressure, and particularly whenspecificity within the genome is less critical, primers as short aseight to ten nucleotides can be used.

Ligase Chain Reaction (LCR). In LCR, the two primers are directed to thesame strand, and are contiguous. If a target strand is present in theDNA to be tested, both primers hybridize to adjacent sequences on thetarget. A DNA ligase is added to join the strands. The ligated productis dissociated by an increase in temperature, and the process repeatsafter cooling. The presence of ligated DNA is detected by any of severalmeans (e.g., molecular weight or fluorescence energy transfer)

Essentially all of the advantages of pressure cycling in the PCR applyto the LCR. The increased selectivity for accurately matched probes isparticularly critical in LCR, since it is less sensitive than the PCR tosingle-base substitutions and deletions, especially away from theligation site.

Other Amplification Methods. There are now over a dozen other methods ofnucleic acid amplification. Most of these require ahybridization/dehybridization cycle. The use of pressure cycling as areplacement for temperature cycling will be feasible in any suchamplification reaction.

Without further elaboration, it is believed that one skilled in the artcan, based on the above disclosure and the examples below, utilize thepresent invention at its fullest extent. The following examples are tobe construed as merely illustrative of how one skilled in the art canpractice the invention and are not limitative of the remainder of thedisclosure in any way. Any publications cited in this disclosure arehereby incorporated by reference.

EXAMPLE 1

Hybridization under High Pressure

The purpose of this example is to show an increased discriminationbetween matched and mismatched hybridizations when the hybridization isperformed at a pressure above ambient pressure.

A known sequence of single-stranded DNA is immobilized on glass usingstandard techniques. For example, the DNA is synthesized so as to have aterminal amine, which is reacted with groups covalently bound to theglass. Two probes are made, each twelve base-pairs long andcomplementary to a region of the immobilized DNA. A mismatch error isintroduced during synthesis into one of the two probes. Each probe isalso fluorescently labeled during synthesis.

Buffer (pH, salt) and temperature conditions are determined at which thematched probe is bound to the slide and the mismatched probe is not.(These can be estimated by known methods, since the sequences are known,and can be determined empirically.) The temperature is selected to bethe lowest temperature at which the matched probe is bound and themismatched probe is not. Then the temperature increase needed todissociate the bound matched probe is determined.

The experiment is repeated, using the same probes and slides, at highpressure, for example 10,000 psi. Both probes can be found to be bound.

The buffer is then changed by reduction of its ionic strength until themismatched probe is not bound and the matched probe is bound at ambientpressure. Then the temperature is increased in increments, until thematched probe is no longer bound, and the required temperature increaseis determined. This increase can be found to be greater than thetemperature increase required at ambient pressure.

EXAMPLE 2

Stringent Washing of Hybrids under High Pressure

The purpose of this example is to show an increased discriminationbetween matched and mismatched hybridizations when the hybridizationreaction is washed under high pressure.

Slides are established in a flow cell for efficient washing withmeasurable amounts of buffer. Using the conditions as established inExample 1, the probes are bound to the slide at high pressure at atemperature and buffer concentration sufficient to allow the mismatchedprobe to be bound. Then the pressure is reduced to one known to allowdissociation of the mismatched probe, and the amount of buffer requiredto elute the mismatched probe is determined. The experiment is repeatedat atmospheric pressure, but the buffer concentration is changed toallow dissociation of the mismatched probe. The required volume isdetermined. The dissociation volume can be greater at lower pressure.

EXAMPLE 3

Measurement of Melting Temperature Differences for Matched vs.Mismatched Oligonucleotide Hybrids as a Function of Pressure

An immobilized DNA array containing DNA of various sequences isconstructed by standard methods described in the literature. A mixtureof probes with various degrees of matching to portions of the array issubjected to the manipulations described in Examples 1 and 2 above. Itis observed that the discrimination among probes is significantlyimproved when the entire procedure is conducted at a constant highpressure and is further improved by variation of the stringency byalteration of the pressure (e.g., at high pressure) compared toalteration of the buffer composition or the temperature.

EXAMPLE 4

Measurement of Pressure Effect on Temperature-Jump Relaxation Rate

Double stranded DNA of known sequence is denatured at atmosphericpressure and at elevated temperature by a temperature jump. Denaturationis observed by a downward shift in UV absorption of the nucleic acids.It is found that the rate of absorbance change is faster when thedenaturation occurs at higher pressure. dsDNA with a single mismatch isfound to dissociate with a similar rate, with a wider difference in thecritical temperature compared to matched DNA.

EXAMPLE 5

Electric Field Modulation of Hybridization Under High Pressure

Changes in electric field can also be used to affect hybridizationconditions in conjunction with high pressure. Suitable procedures anddevices are described in U.S. Pat. Nos. 5,632,957 and 5,605,662. Suchprocedures and devices include a system for performing molecularbiological diagnosis, analysis and multi-step and multiplex reactionsutilizing a self-addressable, self-assembling microelectronic system foractively carrying out controlled reactions in microscopic formats. Thesereactions include most molecular biological procedures, such as nucleicacid hybridization, antibody/antigen reaction, and clinical diagnostics.Multi-step combinatorial biopolymer synthesis may be performed. Acontroller interfaces with a user via input/output devices, preferablyincluding a graphical display. Independent electronic control isachieved for the individual microlocations. The controller interfaceswith a power supply and interface, the interface providing selectiveconnection to the microlocations, polarity reversal, and optionallyselective potential or current levels to individual electrodes. A systemfor performing sample preparation, hybridization and detection and dataanalysis integrates multiple steps within a combined system. Chargedmaterials are transported preferably via free field electrophoresis. DNAcomplexity reduction is achieved preferably by binding of DNA to asupport, followed by cleaving unbound materials, such as by restrictionenzymes, followed by transport of the cleaved DNA fragments. Active,programmable matrix devices are formed in a variety of formats,including a square matrix pattern with fanned out electricalconnections, an array having electrical connections and optionallyoptical connections from beneath the specific microlocations.

EXAMPLE 6

Hyperbaric Amplification of DNA

Preparation of reagents: Reagents and control template DNA (531-bp humanGADPH insert contained within pAMP1 UDG cloning vector) from a DNAamplification kit (Cat. # 10200-012, Life Technologies, Gaithersburg,MD) is added to a sterile 0.5 ml amplification tube placed on ice toachieve a volume of 99 μl. One μl of Taq DNA polymerase (5 units) isadded to the tube. The contents of the tube are mixed, then centrifugedbriefly. A 50 μl aliquot of the reaction mixture is removed from theamplification tube and inserted into a sterile polypropylene capsuleplaced on ice. The 50 μl sample remaining in the amplification tube(Sample A), and the 50 μl sample in the polypropylene capsule (Sample B)are both overlaid with two drops of silicone oil.

DNA amplification using a thermocycling procedure: Sample A is placed ina thermocycler (Techne, Princeton, N.J.) set at 80° C. After 5 minutesat 80° C., Sample A is subjected to 30 cycles of amplification. Eachamplification cycle is comprised of a DNA denaturing step at 940C for 30seconds, a DNA annealing step at 60° C. for 75 seconds, and a DNAextension step at 72° C. for 2 minutes. At the end of the 30 cycles, thetemperature is maintained at 72° C. for 10 minutes. The total elapsedtime for the entire DNA amplification procedure is approximately 4hours. The temperature is then maintained at 4° C. until an aliquot ofthe sample is removed for analysis by agarose gel electrophoresis, asdescribed below.

DNA amplification using a hyperbaric cycling procedure: Sample B wasplaced in a the reaction chamber of a hyperbaric cycling reactor(BioSeq, Woburn, Mass.) set at 94° C. After 5 minutes at 94° C., SampleB was subjected to 30 cycles of amplification. Each amplification cycleis comprised of a DNA denaturing step at 94° C. for 30 seconds atatmospheric pressure, a DNA annealing step at 94° C. for 1 minute at80,000 psi, and a DNA extension step at 94° C. for 2 minutes at 80,000psi. At the end of the 30 cycles, the temperature is lowered to 72° C.for 10 minutes. The total elapsed time for the entire DNA amplificationprocedure is approximately 2 hours. The temperature is then maintainedat 4° C. until an aliquot of the sample is removed, at the same time asSample A, for analysis by agarose gel electrophoresis, as describedbelow.

Agarose gel analysis of the amplification products: One μl aliquots ofSample A and Sample B are mixed with 9 μl of an agarose gel containingethidium bromide loading buffer. The samples are then applied to a 1.0%agarose mini-gel, and electrophoresed at 100 V for 40 minutes. DNA inthe samples is quantified by UV illumination and photography. Bothsamples contained approximately 100 ng of DNA per 1 μl of sample.

What is claimed is:
 1. A method of hybridizing a first nucleic acid to asecond nucleic acid at least partially complementary to the firstnucleic acid, the method comprising: (1) providing a sample vessel andpressure controller for the vessel; and (2) contacting the first andsecond nucleic acids within the vessel at a pressure above ambientpressure that is effective to increase hybridization specificity betweenthe first and second nucleic acids.
 2. The method of claim 1, furtherproviding a nucleic acid polymerase and at least one nucleotidetriphosphate and wherein the first nucleic acid has a 3′ terminalnucleotide that hybridizes to an internal nucleotide in the secondnucleic acid, the first nucleic acid capable of being extended at leastone nucleotide by the polymerase using the second nucleic acid as atemplate.
 3. The method of claim 2, further comprising cycling pressurein the vessel between a first higher pressure at which the first andsecond nucleic acid are hybridized and a second lower pressure at whichthe first and second nucleic acid are denatured.
 4. The method of claim3, further comprising providing a temperature control for the samplevessel, and cycling the temperature between a lower temperature and ahigher temperature, such that the first and second nucleic acidshybridize at the first pressure and lower temperature, and such that thefirst and second nucleic acids denature at the second pressure andhigher temperature.
 5. The method of claim 3, wherein the vessel ismaintained at a constant temperature as the pressure is cycled.
 6. Themethod of claim 1, further comprising washing away unhybridized nucleicacids after increasing the pressure but before decreasing the pressure.7. The method of claim 1, wherein the pressure inside the vessel isincreased to greater than 10,000 psi.
 8. A method of detecting in asample the presence of a nucleic acid that hybridizes to a referencenucleic acid with greater specificity at a first higher pressure than ata second lower pressure, the method comprising: (1) providing a samplevessel and pressure controller for the vessel; and in any order (2)contacting the reference sequence with the sample in the vessel at thefirst pressure; (3) contacting the reference sequence with the sample inthe pressure vessel at the second pressure; and (4) detecting thepresence of a nucleic acid that hybridizes to the reference nucleic acidwith greater specificity at the first pressure than at the secondpressure.
 9. The method of claim 8, wherein the reference sequence isfirst contacted with the sample and hybridization is detected, and thenthe pressure is lowered and the absence of hybridization is detected.10. A method of discriminating between a first nucleic acid and a secondnucleic acid that is different from the first nucleic acid, the methodcomprising, (1) providing a sample vessel and pressure controller forthe vessel; (2) maintaining the vessel at a constant pressure; (3)providing the first and second nucleic acid and a reference nucleic acidin the vessel under conditions that do not allow either the first or thesecond nucleic acid to hybridize to the reference nucleic acid; (4)perturbing at least one condition to establish conditions that permitthe first nucleic acid to form a complex with the reference nucleic acidat equilibrium and to permit the second nucleic acid to form a complexwith the reference nucleic acid at equilibrium; and (5) comparing thetime necessary to achieve equilibrium hybridization between the firstnucleic acid and the reference nucleic acid with the time necessary toachieve equilibrium hybridization between the second nucleic acid andthe reference nucleic acid, wherein the difference indicates therelative difference in sequence between the first and the second nucleicacids.
 11. The method of claim 10, wherein the perturbation is a changein temperature inside the vessel.
 12. The method of claim 10, whereinthe perturbation is a change in an electric field inside the vessel. 13.The method of claim 10, wherein the sample vessel is maintained at apressure of at least 10,000 psi.
 14. A method of discriminating betweena first nucleic acid and a second nucleic acid that is different fromthe first nucleic acid, the method comprising, (1) providing a samplevessel and pressure controller for the vessel; (2) providing the firstand second nucleic acid and a reference nucleic acid in the vessel undera first pressure that does not allow either the first or the secondnucleic acid to hybridize to the reference nucleic acid; (3) perturbingthe pressure to establish conditions that permit the first nucleic acidto form a complex with the reference nucleic acid at equilibrium and topermit the second nucleic acid to form a complex with the referencenucleic acid at equilibrium; and (4) comparing the time necessary toachieve equilibrium hybridization between the first nucleic acid and thereference nucleic acid with the time necessary to achieve equilibriumhybridization between the second nucleic acid and the reference nucleicacid, wherein the difference indicates the relative difference insequence between the first and the second nucleic acids.
 15. The methodof claim 14, wherein the sample vessel is maintained at a pressure of atleast 10,000 psi.
 16. The method of claim 4, wherein a portion of thesecond nucleic acid is amplified.